Troponin (Tn) is the calcium-sensing protein of striated muscle thin filaments. Its subunits are biomarkers for disease as mutations of them are the cause of disease. Understanding how Tn interacts with the thin filament is essential in understanding its involvement in disease. Calcium and strong crossbridges induce transitions in the position of Tm on the thin filaments involved in activation of contraction. The strong crossbridge transduces a signal back to Tn to increase its apparent affinity for calcium, but how this occurs is unknown. Strong crossbridges are more important in signal transduction in cardiac than skeletal muscle. How Tn/ thin filament interactions differ between these striated tissues is unknown. Comparative protein exchange and binding studies of Tn or its subunits within and to cardiac and skeletal myofibrils, FRET, and crosslinking studies within Tn in cardiac and skeletal myofibrils are used to determine the Tn's interactions and how they influence activation of contraction. Our first hypothesis is that calcium and strong crossbridges dramatically increase Tn dissociation rate from thin filaments. Our second hypothesis is that the calcium influence on Tn and Tn subunit interactions is less in cardiac than skeletal myofibrils, making the strong crossbridge a more critical component of activation in cardiac muscle. Our third hypothesis is that signal transduction from the strong crossbridge to Tn involves a crossbridge-dependent dissociation of part of Tnl. We will test these by the following Specific Aims: 1) Determine the influence of calcium and strong crossbridges on Tn dissociation rate in cardiac and skeletal myofibrils. 2) Define Tn subunits responsible for the calcium and strong crossbridge effects upon Tn dissociation rate and on regulatory interactions between TnC and Tnl. 3) Determine how designed and disease-associated mutant Tn subunits alter Tn subunit interactions and calcium activation of force in single myofibrils.